An optimized HPLC-MS/MS assay for uracil and dihydrouracil in plasma: Improving dihydropyrimidine dehydrogenase phenotyping for individualized fluoropyrimidine treatment

Dihydropyrimidine dehydrogenase (DPD) is a key phase I drug-metabolizing enzyme responsible for catabolizing fluoropyrimidine chemotherapeutics such as 5-fluorouracil, capecitabine and tegafur. Its activity shows marked interindividual variability due to both genetic polymorphisms and environmental factors. Reduced DPD activity results in excessive fluoropyrimidine exposure, potentially causing severe and life-threatening toxicities. Although DPD testing is recommended before initiating fluoropyrimidine therapy, no single analytical strategy has been universally adopted, leading to substantial variability in clinical practice. While genotyping offers high specificity, phenotyping provides greater sensitivity by capturing both genetic and non-genetic influences on enzyme activity. In this study, we developed and validated an HPLC-MS/MS assay for the simultaneous quantification of endogenous uracil and dihydrouracil in human plasma, widely used endogenous biomarkers for DPD phenotyping. Sample preparation involved extraction with lipid removal, followed by chromatographic separation on porous graphitic carbon (100 × 2.1 mm, 5 μm; Hypercarb™, Thermo Scientific). Validation was performed according to international guidelines, demonstrating appropriate selectivity, sensitivity, linearity, accuracy, precision, carry-over, and stability. The method was applied to 28 human plasma samples, with uracil concentrations compared against an external laboratory using an independent LC-MS/MS platform. Deming regression showed no significant constant bias at the 95% confidence level, although underestimation occurred at higher concentrations (y = 0.63× + 4.86). Bland-Altman analysis indicated a small mean difference of -0.02 ng/mL but wide limits of agreement (-9.61 to +9.57 ng/mL), highlighting the impact of analytical variability near clinical decision thresholds. Further studies are warranted to evaluate the clinical utility of this approach in larger cohorts and to assess its correlation and complementarity with DPD genotyping and clinical outcomes. This method represents a valuable analytical tool to support personalized fluoropyrimidine-based chemotherapy in patients with solid tumors.